The present invention is directed to apparatus for providing electrical stimulation to cultures of myocytes, which are contractile biological cells, such as muscle and heart cells.
Much of basic cardiac research is being done at the cellular level with many researchers using isolated cardiac myocytes in culture as their experimental material. Whereas non-beating myocytes in culture de-differentiate and atrophy over time, it has been shown that electrically stimulated, continuously beating myocytes maintain their morphology and contractility and retain their normal protein synthesis levels. The useful life span of the myocytes in culture is thus increased by many days. By proposing the design of a device for providing electrical stimulation “pacing” to myocyte cultures, this apparatus provides a unique but basic tool that will make chronic pacing of cultured myocytes an affordable and convenient technique to the cardiac research community.
The intense interest in understanding the physiology and patho-physiology of the heart has led to a substantial increase in the number of laboratories that study the myocardium at the cellular level using isolated cardiac myocytes. Culturing cardiac myocytes in serum-free, well-defined media is the method of choice for studying cell physiology and metabolism under tightly controlled experimental conditions. Developed in the seventies and eighties, the applications for myocyte culture have grown explosively and Myocyte culture is now a mainstream technique.
There exist two primary needs for a Myocyte culture pacing apparatus of the type described herein. The first is the ability for long term maintenance of cultured cells over a period of several weeks. Second, a Myocyte culture pacing apparatus is needed to allow laboratories to scale up the quantity of myocytes cultured. Many experiments require a long-term, stable, preparation of myocytes. These include myofilament gene transfer with the use of adeno-viruses, studies on the regulation of protein turnover, protein incorporation, or the hypertrophic effect of a specific drug or treatment,
It has long been known that adult cardiac myocytes can be cultured for more than a month in the presence of serum. Unfortunately, serum is not a very well defined medium. Serum also contains many trophic factors, which is undesirable when studying processes related to growth. This has led most laboratories to perform their long-term studies with embryonic or neonatal cells, which can be cultured in serum-free media. However, extrapolation from embryonic or neonatal myocytes to the fully differentiated myocardium is problematic. Embryonic or neonatal cells so strongly differ in morphology from fully differentiated cells that it is not possible to measure important functional parameters such as contractility. Also, the expression of ion channels and contractile protein isoforms changes during cell development.
The alternative is the culture of adult myocytes in serum free media. Much progress has been made over the past years and myocytes can be kept viable now for several weeks. Problems that remain are low survival rates and atrophy. Atrophy results in a loss of regular myofibrilar structure which manifests itself as a disappearance of the striation pattern. Therefore a substantial need exists for alternative methods of long-term culture that do retain the typical characteristics of adult cardiac myocytes. Chronic pacing has emerged as a possible solution for the above problems.
Adult cardiac myocytes that are cultured without the presence of growth factors require mechanical stimuli to prevent atrophy. Pacing the cell with electrical pulses provides such a stimulus. Long term electrical stimulation of myocytes plated in serum-free media has been shown to prolong the viability of the cells, retain cell phenotype, and keep levels of protein synthesis comparable to that of the intact heart. Contractility is preserved and important functional parameters such as amplitude of contraction and peak calcium current are increased compared to non-paced cells in culture. Photomicrographs were made of a cell that was beating at 1 Hz and a non-paced quiescent myocyte, from the same animal, after a week of culture. The paced myocyte retained its size and still had a clearly striated myofibrilar content, where the quiescent cell had a rounded appearance and was seen to be losing its striation pattern. The underlying process was found to be that in the quiescent cell the rate of protein synthesis was reduced, leading to a loss in total protein, whereas chronic pacing lead to a recovery and subsequent steady state level of protein synthesis.
The ideal stimulator for long term pacing has several qualities. One of the key issues is that cell culture takes place in a closed system. Byproducts of the electrical field such as the generation of protons or oxygen radicals, and heat production have to be minimized. Several strategies will be applied in the Myocyte culture pacing apparatus.
Heat production and oxidation products are largely a function of the properties of the electrode which have been redesigned in the present invention. Proton accumulation due to electrolysis is avoided by reversing the polarity between pulses, a solution that is not available on any commercial stimulators. Another potential problem is current leakage between pulses, while this is not an issue with continuous perfusion, it is unacceptable in a closed system because it will heat up the preparation. These problems are addressed in the present design.
Experiments using electrical stimulation are normally done using continuous superfusion that serves among other things to remove the oxidative products generated during (and between) pacing stimuli. Continuous superfusion can not be done conveniently in the cell culture incubator. Consequently, special care must be taken to avoid the generation of oxidative products (principally protons and oxygen radicals) in the culture-pacer design. Small Platinum electrodes are typically used for field stimulation during cellular experimentation. However, at the high voltages used in the present Myocyte culture pacing apparatus small sized platinum electrodes will generate too many oxidation products toxic to the cells. The present electrode unit preferably uses large size chemically and biologically inert carbon electrodes which do not generate deleterious oxidation products. The electrodes can also be cleaned and autoclaved for repeated use.
Cultured myocytes can be used for protein assays or for quantitative analysis of other cellular products (e.g. mRNA). Such experiments require a large number of cells due to the typically minute amount of material present in a single cell. These quantities are then multiplied by the number of treatments or time-points the researcher decides to compare. From an economical and statistical point of view, it is desirable to make as many valid measurements as possible from one single animal or at least within one experiment. One great difficulty the researcher is presented with is the large power required from the stimulator to scale up the quantity of paced cells. This points to the need for a pacing instrument that can provide many high voltages output channels and the means to distribute them to myocytes in culture dishes.
No electronic stimulator that is currently available is suitable for pacing large quantities of cells, or cells in long-term culture. The function of a standard cell stimulator is to provide electrical stimuli to a wide range of physiological applications, from muscle fibers to neurons. To cater to such a variety of applications, standard stimulators usually support the generation of quite sophisticated voltage waveforms. The result of this admirable flexibility is a user interface which tends to be more complex than necessary for the simple waveforms required from the present myocyte culture pacing apparatus. A second common assumption in the design of standard stimulators is that they will be used on small preparations. The maximum voltage and current throughput available from these devices simply is not sufficient for field stimulation of whole culture flasks.
The present myocyte culture pacing apparatus satisfies the following specific aims:
1. Provide adjustable output voltage and current in order to work with standard culture dishes or flasks, and different preparations.
2. Provide high voltage, high current throughput to allow multiple preparations or large volumes to be paced
3. Minimize the oxidative products that naturally accumulate during prolonged stimulation in a “closed system”. This is achieved by the use of appropriate electrode material, alternating pulse polarity and employing circuitry that guarantees no current leakage between pulses.
4. The present myocyte culture pacing apparatus can serve as a stand-alone, programmable instrument that, once programmed, requires no monitoring.
5. the present apparatus has simple and straightforward user controls to minimize training time and protocol input errors.
Currently no commercial device is available for chronic pacing of cardiac myocytes that meets these aims. The present device will therefore be an enhancement for laboratories worldwide. The present device enables greater flexibility and economy in the use of cultured cardiac myocytes for heart studies.
The present invention is directed to myocyte culture pacing apparatus operating in conjunction with a standard culture dish that permits long term maintenance of cultured cells over a period of several weeks The device includes a base unit for receiving the culture dish, and an electrode unit for placement atop the culture dish which can receive the cover of the culture dish. The electrode unit has a first and a second planar conductive electrodes extending into each well of the culture dish. The electrodes are planar and extend across the width of the well and are connected to electrical connector for connection to an external electronic unit, which supplies the a variety of electrical pulse regimes to pace the myocyte cultures. The electronic unit permits the type, frequency, duration, voltage and the current of the pulses to be varied in accordance with the desired pacing regime.